The overall aim of this study is to determine the phenotype, function, and role of CD4+CD25+ regulatory T cells (Treg cells) in HIV infected and uninfected humans. In the first specific aim we will sort Treg cells based upon the expression of CD4 and CD25, and then use additional markers to further refine the phenotype of functional Treg cells using multi-color flow cytometry. The phenotypic characterization will be combined with functional experiments, where the sorted subpopulations of Treg cells will be assayed for their suppressive capacity in vitro. In a cross sectional study, we will quantify the number, frequency, and suppressive activity of Treg cells in PBMC obtained from 30 healthy individuals, and from Gut associated lymphoid tissue (GALT) in 6 volunteers. In a longitudinal study on 6 donors on a bi-monthly basis over two years we will measure the number, frequency and activity of the Treg cell population under normal conditions. We will also characterize the mechanism of Treg cell mediated suppression. In the second specific aim we will determine the impact of removal of Treg cells on the avidity, breadth, phenotype and function of HIV specific responding T cells, and also ascertain whether HIV specific Treg cells can be induced during HIV infection. We hypothesize that the functional avidity of a responsive T cell population will change after Treg cell removal, and we will test this using both HIV and CMV specific T cells. We further hypothesize that when eg cells are removed the breadth of the HIV specific T cell response will be larger and new epitopic regions will be revealed. We hypothesize that Treg cells can be induced by HIV infection in the periphery and that they suppress HIV specific T cell responses early in infection. The third specific aim of this study is to determine the impact of Treg cell frequency, number and function on HIV infection in peripheral blood and in GALT. We predict that subjects with a strong suppression of HIV specific T cell responses, and a weak suppression of non-HIV specific T cell responses, will have higher viral load and greater general immune activation. We will also ascertain the impact of highly active antiretroviral therapy (HAART) on Treg cells, and the effect of treatment interruptions on Treg cell functions. The goal of this proposal is to generate data to determine the importance of Treg cells in HIV infection. Manipulation of Treg cells could be critical for vaccine strategies and for therapeutic interventions.